Adhesive N,O-carboxymethylchitosan coatings which inhibit attachment of substrate-dependent cells and proteins

ABSTRACT

The present invention relates to a method of inhibiting cellular and protein attachment to substrates by applying a composition containing an effective amount of adherent N,O-carboxymethylchitosan to a substrate with such that cellular and protein attachment are prevented or greatly reduced.

REFERENCE TO RELATED APPLICATIONS

This application is a Divisional which claims the priority of U.S.patent application Ser. No. 10/672,072, filed Sep. 25, 2003 now U.S.Pat. No. 6,894,035, which is a Continuation in Part of U.S. patentapplication Ser. No. 09/315,480, filed May 20, 1999, now U.S. Pat. No.6,645,947B1, the entirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The attachment of cells and proteins to substrates is a well-knownproblem that has presented itself in a number of contexts. For example,in cell cultures to produce antibodies, fibroblasts attach toextracellular matrix proteins bound to the tissue culture substrate.Similarly, in urinary catheters, bacterial cells attach to the walls ofthe catheter; in arterial catheters, platelets attach to the tip of thecatheter; and in contact lenses, proteins coat the surfaces of thelenses.

Various bioadhesives are known in the art. U.S. Pat. No. 4,615,697,issued to Robinson et al., defines a bioadhesive as a material thatrequires a force of at least about 50 dynes/cm² to separate two adhered,freshly excised pieces of rabbit stomach, following the proceduredisclosed therein. The bioadhesive disclosed in Robinson et al. is awater-swellable, but water insoluble, fibrous, cross-linkedcarboxy-functional polymer.

Various attempts to ameliorate the problem of attachment of cells andproteins to substrates have been employed, but none have been found tobe satisfactory. It would be desirable to solve this problem using abiocompatible substance that is adherent to substrates and inhibitscellular and protein attachment.

Certain cells, such as macrophages and fibroblasts, are referred to as“substrate-dependent cells” because they are active and proliferate onlywhen attached to a surface or substrate. The attachment occurs via afamily of proteins (“attachment molecules or proteins”), such asvitronectin and fibrinectin, which are found in the extracellularmatrix. A surface that is coated with a material that is stronglyadhesive may inhibit the attachment of substrate dependent cells byblocking attachment of extracellular matrix proteins. Hence, adhesivematerials, as described herein, are useful in compositions or can formdevices that inhibit the attachment of certain proteins and certaintypes of cells.

SUMMARY OF THE INVENTION

The present invention features a method of inhibiting cellularattachment to substrates. The invention is based, in part, on thediscovery of adherent coatings of N,O-carboxymethylchitosan (“NOCC”),and in particular that adherent coatings of NOCC may be applied tovarious substrates, such as mammalian tissue, so as to inhibitattachment of other cells, such as substrate dependent cells. Further,it has been discovered that these adherent coatings of NOCC may be usedin other areas where inhibition of cell or protein attachment isdesirable, such as in the preparation of cell populations, on medicaldevices, and with cell-based products. The invention also hasapplication to the inhibition of the attachment of proteins to surfaces.

The present invention provides a composition that is adherent to avariety of synthetic materials and mammalian tissues. The presentinvention also provides a method of inhibiting cellular and proteinattachment to a substrate by applying adherent coatings of NOCC to thesubstrate such that the attachment of cells and proteins is inhibited.The amount of adherent NOCC in the composition should be effective toinhibit the attachment of substrate-dependent cells, preferably in aconcentration of 0.05–5%(w/v), most preferably in a concentration of0.1–2.5%(w/v).

In one embodiment, the invention provides a composition and method ofinhibiting attachment of substrate-dependent cells to a substrate byapplying a composition containing adherent NOCC to a substrate such thatattachment of substrate-dependent cells is inhibited. The method may beapplied to inhibit substrate-dependent cell attachment to mammaliantissue, medical devices, fermentation units, bioreactors and solidsupports. In preferred embodiments, the substrate-dependent cells whichare inhibited include fibroblasts, macrophages, epithelial cells, andendothelial cells.

In another embodiment, the invention provides a composition and methodof inhibiting attachment of proteins to a substrate by applying acomposition containing adherent NOCC to a substrate such that attachmentof proteinaceous material is inhibited. The method may be applied toinhibit protein attachment to contact lenses, medical devices,fermentation units, bioreactors and solid supports.

In another embodiment, the invention may be used in a method ofobtaining a population of cells, e.g., mammalian cells, by supplementingculture media with adherent NOCC, growing the population of cells in thesupplemented media, and allowing the cells to grow or differentiate,such that substrate-dependent cells do not proliferate within the cellpopulation.

In another embodiment, the invention provides a method of obtainingcells suitable for use in protein or antibody production bysupplementing culture media with adherent NOCC and growing the cells inthe supplemented media, such that intercellular attachment (or clumping)within the cell population is inhibited and production of proteins orantibodies is enhanced.

In yet another embodiment, the invention provides a method of inhibitingattachment of inflammatory cells and platelets to a medical device bycoating said device with a composition containing adherent NOCC, suchthat platelet or inflammatory cell attachment to the medical device isinhibited. In preferred embodiments, the internal medical device iseither a stent or shunt. In other preferred embodiments, theinflammatory cell includes fibroblasts, macrophages, and monocytes.

In still another embodiment, the invention includes a method ofinhibiting fibroblast attachment in a cell-based product in contact witha solid support by introducing adherent NOCC into the cell based productsuch that fibroblast attachment is inhibited.

In another embodiment, the invention provides a composition and methodof delivering drugs, proteins, and other therapeutic agents from anadhesive device or composition that is adherent to soft (mucosal ornon-mucosal) tissue or hard tissue. In preferred embodiments, theadherent delivery device can be used as a buccal, oral, vaginal,inhalant, or the like delivery system. The device can be in a variety offorms including solutions, creams, pellets, particles, beads, gels, andpastes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the apparatus used in Example 1.

FIG. 2 is a bar graph showing the results of Example 1.

FIG. 3 is a schematic of the procedure used in Example 2.

FIG. 4 is graph showing the total volume of ¹²⁵I-NOCC adhered to ratfemur, as calculated using Equation 1.

FIG. 5 is graph showing the total volume of ¹²⁵I-NOCC adhered to ratfemur, as calculated using Equation 3.

FIG. 6 shows the morphological difference between fibroblasts grown inthe absence of NOCC (6 a) and fibroblasts grown in the presence of NOCC(6 b).

FIG. 7 is a bar graph showing a comparison of adherence by fibroblastsgrown in the presence and absence of NOCC, under various pre-coatingconditions.

FIG. 8 is a bar graph showing a comparison of adherence by fibroblastsgrown in the presence and absence of NOCC, under various pre-coatingconditions.

FIG. 9 is a bar graph showing a comparison of adherence by fibroblastsgrown in the presence and absence of NOCC, as determined by ⁵¹Cr releaseassay.

FIG. 10 is a bar graph showing a comparison of adherence by fibroblastsgrown in the presence and absence of NOCC, as determined by ⁵¹Cr releaseassay.

FIG. 11 is a bar graph showing a comparison of adherence by fibroblastsgrown in the presence and absence of NOCC under various pre-coatingconditions, as determined by ⁵¹Cr release assay.

FIG. 12 is a bar graph showing a comparison of adherence by fibroblastsgrown in plates that were pre-coated with and without NOCC, asdetermined by using cells labeled with tritiated thymidine.

FIG. 13 is a bar graph showing a comparison of adherence by epithielialcells grown in plates that were pre-coated with and without NOCC, asdetermined by using cells labeled with tritiated thymidine.

FIG. 14 is a bar graph showing a comparison of adherence by macrophagesgrown in plates that were pre-coated with and without NOCC, asdetermined by using cells labeled with tritiated thymidine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the inhibition of cellular and proteinattachment to various substrates. The method of the invention uses anadherent coating of N,O-carboxymethylchitosan (“NOCC”) which providesunexpected benefits in inhibiting cellular and protein attachment.

NOCC is a derivative of chitin, which is found in the shells ofcrustaceans and many insects. Chitin and its derivatives are normallybiocompatible, naturally resorbed by the body, and have previously beenused for sustained drug release, bone induction and hemostasis (Chandyand Sharma, Biomat. Art. Cells & Immob. Biotech. 19:745–760 (1991);Klokkevold, P. et al., J. Oral Maxillofac. Sur. 50:41–45 (1992)). Due toits prevalence, chitin may be obtained relatively cheaply, largely fromwaste products. As disclosed in U.S. Pat. No. 4,619,995, issued toHayes, the entire contents of which are hereby incorporated byreference, NOCC has carboxymethyl substituents on some of both the aminoand primary hydroxyl sites of the glucosamine units of the chitosanstructure. NOCC may be used in an uncrosslinked form as a solution ormay be cross-linked or complexed into a stable gel. Because of itsadvantageous physical properties, and its relative low cost, NOCCpresents advantageous properties for use in inhibiting cellular andprotein attachment.

Definitions

The term “inhibit,” or any form thereof, is defined in its broadestsense and includes minimize, prevent, repress, suppress, curb,constrain, restrict and the like.

The terms “adherent NOCC” or “an adherent coating of NOCC” mean acoating or composition of NOCC that exhibits an adhesion between freshlyexcised tissue of at least about 100 dynes/cm², using the proceduredescribed in Example 1.

The term “substrate” refers to any object to which cells can attach.Examples of substrates include, without limitation, mammalian tissue(including both hard tissue, such as bone, and soft tissue, such asmucosal and non-mucosal tissue), non-mammalian tissue, mammalian andnon-mammalian cells (including both eukaryotic and prokaryoticorganisms), medical devices, fermentation units, bioreactors, and solidsupports, such as cell culture plates.

The term “substrate-dependent cells” means cells that are only activewhen attached to a substrate. Examples of substrate dependent cellsinclude, without limitation, fibroblasts, macrophages, epithelial cells,somatic cells, and endothelial cells.

The term “medical device” means any device which is implanted in thebody for medical reasons or which has a portion of the device extendinginto the body (like a catheter) as well as devices which provide amedical benefit when attached to, or are in contact with, the body.Examples of medical devices include, without limitation, catheters,contact lenses, stents, shunts, breast implants and pacemakers.

The term “inflammatory cell” means a cell involved in the non-specificimmune response to any type of body injury. Examples of inflammatorycells include, without limitation, fibroblasts, macrophages,eosinophils, neutrophils, monocytes and lymphocytes.

The term “cell-based product” means any product that contains cell.Examples of cell-based products include, without limitation, blood,plasma, aliquots of cell cultures, and the like.

The invention provides a method of inhibiting attachment ofsubstrate-dependent cells or proteins to a substrate by applying acomposition containing adherent NOCC to a substrate such that attachmentof the substrate-dependent cells or protein is inhibited. In preferredembodiments, the method is applied to inhibit substrate-dependent cellattachment to mammalian tissue, medical devices, fermentation units,bioreactors and solid supports. In preferred embodiments, thesubstrate-dependent cells which are inhibited include fibroblasts,macrophages, epithelial cells, and endothelial cells.

The invention also may be used in a method of obtaining a population ofcells, e.g., mammalian cells, by supplementing culture media withadherent NOCC, growing the population of cells in the supplementedmedia, and allowing the cells to grow or differentiate, such thatsubstrate-dependent cells do not proliferate within the cell population.

The invention further provides a method of increasing the efficiency ofprotein or antibody production by supplementing culture media withadherent NOCC and growing the cells in the supplemented media, such thatintercellular attachment within the cell population is inhibited andproduction of protein or antibodies is enhanced.

The invention also provides a method of inhibiting attachment ofinflammatory cells or proteins to a medical device by coating saiddevice with a composition containing adherent NOCC, such thatinflammatory cell or protein attachment to the medical device isinhibited. In preferred embodiments, the medical device is a catheter, acontact lens, a stent, pacemaker, breast implant, or a shunt. The methodis useful for preventing attachment of a variety of inflammatory cellsincluding fibroblasts, macrophages, monocytes, as well as proteins suchas albumin.

In still another embodiment, the invention includes a method ofinhibiting fibroblast attachment in a cell-based product in contact witha solid support by introducing the cell based product to an adherentcoated solid support such that fibroblast attachment is inhibited.

The adherent NOCC used in the present invention may take many forms. Forexample, adherent NOCC may be used in a solution, a hydrogel, a paste, arehydratable film, cream, foam, or a sponge. These forms are prepared bymethods well known to those of ordinary skill in the art.

The adherent NOCC used in the present invention may be the parentcompound or may be cross-linked. Cross-linked adherent NOCC may beeither covalently cross-linked or ionically cross-linked. Variousmethods of cross-linking NOCC are known in the art and are within thescope of this invention. In addition, the degree to which the adherentNOCC is cross-linked may be optimized for specific applications by oneof ordinary skill without undue experimentation. It has been found thatthe degree of cross-linking is roughly inversely proportional to theadhesiveness of the coating. That is, the greater the degree ofcross-linking of the adherent NOCC, the lesser degree of adherence. Inpreferred embodiments, the degree of cross-linking is less than 1:5(moles cross-linking agent to moles, NOCC monomer), more preferablybetween 1:100 and 1:1000 on a molar basis.

The bioadhesive strength of several adherent NOCCs was compared to thatof polycarbophil, a cross-linked acrylic acid polymer available fromB.F.Goodrich. As more fully described in Example 1, solutions of low andhigh viscosity NOCC were prepared, as well as hydrogels of highviscosity NOCC. The bioadhesive was applied to stomach and cecal tissuesamples and the bioadhesive strength was measured according to amodified version of the procedure disclosed in U.S. Pat. No. 4,615,697,which is hereby incorporated by reference. The transfer of polymer toboth tissue surfaces indicated that the adhesive force of the polymerexceeded the cohesive force. A summary of results appears in Tables 1and 2, and FIG. 2. In preferred embodiments, the bioadhesive strength ofadhesive NOCC coatings of the invention is desirably greater than atleast about 1000 dynes/cm², more preferably greater than at least about2000 dynes/cm², and most preferably greater than at least about 3000dynes/cm².

Both the low viscosity and high viscosity NOCC polymer solutions incitrate buffer behaved similarly to polycarbophil when applied as acoating to the mucosal surface of stomach tissue (Table 1). This wasalso true for similar solutions of NOCC using phosphate buffered salineinstead of citrate buffer as well as non-mucosal, cecal tissue (Table2). It was observed that as NOCC was cross-linked the cohesion of thematerials increased and the adhesion decreased. The loss of adhesion wasdependent on the extent of cross-linking. These findings are likelyattributable to the fact that cross-linking adherent NOCC introducedmore structure into the polymer, which consequently restrictedinteractions with the tissue surface. The cross-linking also joined thepolymer chains together, resulting in increased cohesiveness.

The ability of NOCC to adhere to bone tissue was also studied. Theresults indicate that NOCC adheres to bone tissue (FIG. 5). After thethird wash, 9.5×10⁻³ ±0.002 μL/mm² (or about 0.1 μg NOCC/mm²) of ¹²⁵Ilabeled NOCC remained adhered to the rat femur.

Surprisingly, the adhesive NOCC coatings of the present invention havebeen shown to inhibit cellular attachment of substrate dependent cells.The adherent NOCC coatings of the present invention thus haveapplicability in a multitude of areas. In addition, adherent NOCCcoatings may be applied to either hard or soft mammalian tissue, such asbone or stomach tissue. Alternatively, adherent NOCC coatings may beapplied to non-biological substrates, such as medical devices and solidsupports. Examples of such substrates include stents, shunts, contactlenses, microtiter plates, and cell culture plates.

Typically, fibroblasts in a cell or tissue culture adhere toextracellular matrix (ECM) proteins that are bound to the culturesubstrate (usually plastic). The ECM proteins in culture typically comefrom the culture medium, which is supplemented with serum to providethese proteins as well as other factors necessary for cell growth.Alternatively, if there are no ECM proteins in the culture medium,fibroblasts will secrete their own ECM proteins and adhere to them. Anormal, adhered fibroblast has a very characteristic morphology: itflattens and exhibits cellular appendages or processes extending fromthe cell over the substrate surface, which indicates fibroblastadherence to the substrate (FIG. 6 a).

The present invention takes advantage of the observation thatsubstrate-dependent cells, e.g., fibroblasts, plated in tissue culturemedia in the presence of adherent NOCC coating do not have thecharacteristic morphology and do not exhibit processes indicatingattachment of the cell (FIG. 6 b).

Initial observations of fibroblast morphology in the presence or absenceof adherent NOCC in the medium, revealed that when fibroblasts wereplated in serum-free media without adherent NOCC they consistentlydisplayed an “adherent” morphology, viz. the cells were flattened withcomplex processes. As described more fully in Example 3, fibroblastswere plated on tissue culture plates, either in the presence or absenceof adherent NOCC, under four different coating conditions. Irrespectiveof the coating treatment, approximately 80% of cells observed lookedlike normal cultured fibroblasts in the absence of adherent NOCC. Incontrast, when fibroblasts were plated in the presence of adherent NOCC,the number of cells displaying the adherent morphology was significantlyreduced. In fact, in the instance where no ECM proteins were present, nocells adhered in the presence of adherent NOCC. Where ECM proteins werepresent, some cell adherence was observed, but the adherence wassignificantly less than that which occurred in the absence of adherentNOCC (FIG. 7).

Hyaluronic acid (HA) was also tested in this system, to determinewhether it had similar effects to NOCC. When a similar morphologicalexamination was performed on cells plated in sfRPMI (serum free orprotein free RPMI medium) containing 0.1% HA, it was observed that theHA did not have the same effect on fibroblast morphology (FIG. 8).

Fibroblast adherence was also measured quantitatively, using a ⁵¹Cradhesion assay (the ⁵¹Cr release assay). The results confirmed thatadherent NOCC blocks adhesion of 3T3 fibroblasts to plastic, by morethan 90% using this assay (FIG. 9). This result, taken together with theprevious work, suggests that adherent NOCC adheres to the substrate andinterferes with the deposition of ECM proteins in a competitive manner.

A competitive assay was performed using media supplemented with varyingconcentrations of fetal calf serum (FCS), which contains the ECMproteins of interest, and in the presence or absence of NOCC. Asexpected, it was found that the presence of FCS in the plating mediumreversed the inhibitory effect of adherent NOCC on fibroblast adhesionin a dose dependent manner, where 10% FCS fully restored binding of 3T3to the plates in the presence of NOCC (FIG. 10). There are two possibleexplanations for this effect: 1) adherent NOCC prevents fibroblastadhesion to the ECM proteins which bind to the plate, or 2) adherentNOCC prevents the binding of ECM proteins to the plate in a competitivemanner.

To address these possibilities, tissue culture plates were pre-coatedwith serum-free medium containing varying concentrations of FCS. Thepresence of adherent NOCC did not interfere with the adhesion offibroblasts to the FCS coated plates, which confirmed that adherent NOCCdoes not inhibit adhesion of fibroblasts to ECM proteins alreadydeposited on the plate (FIG. 11). This result was confirmed by coatingtissue culture plates with an adherent NOCC coating. The results (FIG.12) demonstrate that fibroblast adherence to adherent NOCC coatedplastic is eliminated in the presence of FCS. This result supports thehypothesis that adherent NOCC binds to the plastic plate surface andprevents the deposition or attachment of ECM proteins. Thus, adherentNOCC may inhibit cellular attachment by preventing the deposition of ECMproteins rather than by inhibiting the adhesion of fibroblasts to theECM proteins. In the absence of the ECM protein network, fibroblasts areunable to bind to a substrate.

The following, non-limiting examples will further elucidate theinvention.

EXAMPLE 1

In this example, the bioadhesive strength of several adherent NOCCcoating compositions is compared to that of polycarbophil. Polycarbophil(B.F. Goodrich, Akron, Ohio) was prepared as a 4% w/v solution in both0.2M citrate buffer (pH 4.8) and 0.9% saline (pH 6.8). Low viscosity(“LV”) NOCC (240 cps, Brookfield spindle 3, 50–100 rpm) was prepared as4% w/v solution in citrate buffer (pH 5.6). High viscosity (“HV”) NOCC(P78NOCC1) was prepared as 2.5% w/v solution in citrate buffer (pH 5.6).High viscosity NOCC was prepared as 1% and 2.5 % in citrate buffer (pH5.6–5.7), autoclaved and cross-linked (1:500). HV NOCC was also preparedas 2.5% solution in phosphate buffered saline (PBS). Gels were formedfrom 1% HV NOCC by cross-linking (1:100) in PBS and by cross-linking(1:250) in saline following autoclaving.

Both stomach and cecal tissues from Sprague-Dawley rats were harvestedimmediately prior to testing and were kept moist in saline solution.Tissue samples were mounted on circular plastic disks with the innersurfaces of stomach tissues and the outer surfaces of cecal tissuesexposed. Tissue samples were held in place with a suture around the endof the plastic disks. The plastic disks were obtained from the plungersof 3 and 5 ml syringes; the diameters of the disks were 7.0 (surfacearea of 38.5 mm²) and 9.5 mm (surface area of 70.9 mm²), respectively.The tissue holders were attached to a cantilever load cell and to theactuator of an MTS servohydraulic material testing machine (see FIG. 1).

The temperature compensated load cell was wired into a Daytronic 3720Strain Gauge Conditioning Unit in a half bridge configuration. Datacollection was performed using a Macintosh Centris 650 computer equippedwith labVIEW software and a 12 bit NB-MIO-16 data acquisition board. Thecantilever load cell was calibrated over the working range of 0–3 gramsusing a series of proving masses (0.1, 0.23, 0.5, 1 to 3.0 g) verifiedon a Mettler PJ 360 balance. A least squares calibration curve wasdetermined to convert the resulting output from volts to grams force.

The smaller diameter tissue of the pair of fresh tissue samples received30 μl of test material. The software was designed to take a zero readingafter attaching the tissue samples and applying a coating of thebioadhesive. The testing system actuator was then manually advancedusing the displacement potentiometers to bring mating faces intocompression while visually monitoring the resulting load level on thecomputer monitor. The mating faces were allowed to remain compressed ata nominal load of 0.9 g for one minute. The computer then displaced theactuator at a constant rate of 12.0 mm/min, monitoring the distractionforce with time. After failure the computer determined the peakdistraction load and saved the loading curves to a spreadsheet file.

For repeated testing of the same samples, the tissues were scraped withthe side of a syringe needle, rinsed with citrate buffer or water asappropriate and a new aliquot of the same polymer was applied. Freshtissues were used for each different polymer sample; all samples incitrate buffer were tested on stomach tissue and all samples at neutralpH were tested on cecal tissue. All testing was performed in air.

All polymer samples were applied to the smaller surface area tissuesample at a rate of approximately 1 μl per sq.mm. Following distractionof the actuator, the transfer of polymer to both tissue surfacesindicated that the adhesive force of the polymer exceeded the cohesiveforce. For example, polycarbophil was adhesive to both cecal and stomachtissue and required a tensile force of 2300–2800 dynes/cm² to causefailure. The failure was cohesive rather than adhesive since polymer wasobserved on both tissue surfaces after separation. A summary of resultsappears in Tables 1 and 2 and FIG. 2.

Both the low viscosity and high viscosity adherent NOCC polymersolutions in citrate buffer behaved similarly to polycarbophil whenapplied as a coating to the mucosal surface of stomach tissue. Bothadherent NOCC samples failed cohesively and required larger forces toachieve tissue separation than for polycarbophil. However, when highviscosity NOCC solutions were cross-linked to form hydrogels, theybecame more cohesive and failed by detaching from the larger diameterdisk at forces of 85% (1% gel) and 53% (2.5% gel) of that ofpolycarbophil.

The strengths of adhesion to the external surface of the cecum (Table 2)again demonstrated that a solution of NOCC (2.5%-high viscosity) wascomparable to polycarbophil. It was also observed that as adherent NOCCwas cross-linked the cohesion of the materials increased and theadhesion decreased. The loss of adhesion was dependent on the extent ofcross-linking.

It should be noted that polycarbophil measured under the presentconditions exhibited twice the adhesive force as reported in U.S. Pat.No. 4,615,697. This is presumably due to testing in air rather than insolution. For both stomach and cecal tissues, adherent NOCC solutionswere either comparable to or exceeded the performance of polycarbophil:the force required to achieve failure was equal to or larger than thatof polycarbophil and failure was due to cohesion not adhesion.

NOCC hydrogels on both types of tissue were adhesive; however, they weresignificantly less adhesive than materials that were not cross-linked.They demonstrated an adhesive failure rather than cohesive; also it wasobserved that increasing the extent of cross-linking decreased theadhesive force. These findings were not surprising since cross-linkingadherent NOCC introduced more structure into the polymer whichrestricted interactions with the tissue surface and also joined thepolymer chains together resulting in increased cohesiveness.

Another finding was that both the 2.5% high viscosity NOCC solution andthe 1% NOCC gel in citrate were more adhesive than its counterparts inPBS. Without limitation to the present invention, this difference maypossibly be explained by the influence of the citric acid environment.At neutral pH, NOCC exists as an anionic species resulting from thepresence of negatively-charged carboxylate groups (—COO); the freeamines on NOCC are primarily uncharged. By contrast, in acidic citratebuffer (pH 5.6) the amine groups are protonated to formpositively-charged ammonium sites (—NH₃+) which ionically bind citrateions. Such salts are described in U.S. Pat. No. 5,412,084, thedisclosure of which is incorporated herein by reference. Since citratehas 3 carboxylate groups, 2 of which are negatively-charged at pH 5.6,the net result is that NOCC in acidic citrate has an increased number ofcarboxylate groups associated with the polymer and, hence, displays anincreased bioadhesiveness.

TABLE 1 Bioadhesion of NOCC Formulations to Stomach Tissue. Force toSeparate Tensile Failure Tissue Adhesive or Polymer Sample Force (grams)(dynes/sq · mm) Cohesive Failure 4% Polycarbophil 0.901 ± 0.035 2295 ±170 Cohesive 4% LV NOCC 1.007 ± 0.107 2567 ± 270 Cohesive solution 2.5%NOCC(HV) 1.513 3857 Cohesive 1% NOCC gel 0.770 ± 0.280 1961 ± 410Adhesive 2.5% NOCC gel 0.481 1226 Adhesive Notes: Error limits are oneaverage deviation based on 2–3 determination and values without errorlimits result from a single measurement.

TABLE 2 Bioadhesion of NOCC Formulations to Cecal Tissue. Force toSeparate Tensile Failure Tissue Adhesive or Polymer Sample Force (grams)(dynes/sq · mm) Cohesive Failure 4% Polycarbophil 1.113 2837 Cohesive2.5% NOCC 0.992 ± 0.060 2567 ± 140  Cohesive (HV) solution 1% NOCC gel0.302 ± 0.010 770 ± 30  Adhesive (1:100) 1% NOCC gel 0.410 1045 Adhesive(1:250) Notes: Error limits are one average deviation based on 2–3determination and values without error limits result from a singlemeasurement.

EXAMPLE 2

This example illustrates the adherent property of an adherent NOCCcoating of the present invention.

Six female rats were anaesthetized using sodium pentobarbitol (60 mg/kg)and subsequently sacrificed by cervical dislocation. Twelve femurs wereharvested and stripped of connective tissue by sharp dissection. Excessconnective tissue was removed from the rat femur by immersing the ratfemurs in boiling water for thirty minutes. The femurs were then rinsedand air dried.

Each femur was immersed in 1 ml of ¹²⁵I labeled NOCC such that half thesurface area of the femur was in direct contact with the ¹²⁵I NOCCsolution (FIG. 3). The other half of the femur was used to manipulatethe femur. Subsequently, the femur was either placed directly into ascintillation vial and then placed in a γ-counter rack, or the femur wassubjected to a uniform “wash” before being placed into a scintillationvial and the γ-counter rack.

Four groups of three ¹²⁵I NOCC treated femurs were subjected to eitherone wash, two washes, three washes or no washes. A wash consisted of theuniform agitation of the femur in approximately 150 ml of PBS for fiveseconds. Two washes consisted of a wash, removing the femur from PBS forone second, and then repeating a wash. Hence, three washes consisted ofa wash, removal of the femur, a wash, removal of the femur, and one lastwash. The PBS solution was replaced for each group of femurs.

The activity of ¹²⁵I NOCC was evaluated by a Beckman γ-counter. Theamount of ¹²⁵I NOCC adhered to a rat femur was calculated using Equation1, which uses the activity of 1 ml of ¹²⁵I NOCC (7.2×10⁷ CPM) and theactivity of the ¹²⁵I NOCC on the femur, (detected by the γ-counter). Theresults appear in FIG. 4.

Equation  1: $\begin{matrix}{{volume}\mspace{14mu}{of}\mspace{14mu}{\,^{125}I}\mspace{14mu}{NOCC}} \\{{{adhered}\mspace{14mu}{to}\mspace{14mu}{femur}}\;}\end{matrix}\; = {\frac{{{activity}({CPM})}\mspace{14mu}{of}\mspace{14mu}{sample}}{7.2 \times 10^{7}{CPM}} \times 1\mspace{14mu}{mL}}$

Next, the amount of ¹²⁵I NOCC per unit area of the femur was calculated.The surface area that was in direct contact with the ¹²⁵I NOCC solutionwas calculated for one representative rat femur.

Equation  2: $\begin{matrix}{{surface}\mspace{14mu}{area}\mspace{14mu}{in}\mspace{14mu}{direct}} \\{{contact}\mspace{14mu}{with}{\,^{\mspace{14mu} 125}I}\mspace{14mu}{NOCC}}\end{matrix} = {\frac{2\pi\;{rh}}{2} + {\pi\; r^{2}}}$where h=the total height of the femur; r=the radius of the femur

The amount of ¹²⁵I NOCC per unit area of then calculated, using Equation3, by dividing the surface area of the rat femur in direct contact with¹²⁵I NOCC into the amount of ¹²⁵I NOCC adhered to the rat femur. Theresults appear in FIG. 5.

Equation  3: $\begin{matrix}\begin{matrix}{{{\,^{\mspace{14mu} 125}I}\mspace{14mu}{NOCC}}\mspace{31mu}} \\{{{per}\mspace{14mu}{unit}}\mspace{11mu}}\end{matrix} \\{\;{{area}\mspace{14mu}{of}\mspace{14mu}{femur}}}\end{matrix} = \frac{{\mu\; L\mspace{14mu}{of}\mspace{11mu}{\,^{\mspace{14mu} 125}I}\mspace{14mu}{NOCC}\mspace{14mu}{adhered}\mspace{14mu}{to}\mspace{14mu}{femur}}\;}{{surface}\mspace{14mu}{area}\mspace{14mu}{in}\mspace{14mu}{direct}\mspace{14mu}{contact}\mspace{14mu}{with}\mspace{14mu}{\,^{\mspace{14mu} 125}I}\mspace{14mu}{NOCC}}$

The surface area of the rat femur was calculated to be 228 mm²,(radius=2.25 mm and total femur height=30 mm).

Table 3 outlines the number of washes each femur was subjected to, theactivity of ¹²⁵I NOCC, amount of ¹²⁵I NOCC adhered to femur, and theamount of ¹²⁵I NOCC per unit area of femur.

TABLE 3 volume of ¹²⁵I Number of activity of ¹²⁵I volume of ¹²⁵I NOCC(μL)/ femur washes/ NOCC/femur NOCC adhered unit area of number femur(CPM) to femur (μL) femur (mm²) 1 0 2.3 × 10⁶ 31.9 1.4 × 10⁻¹ 2 0 2.7 ×10⁶ 37.5 1.6 × 10⁻¹ 3 0 2.9 × 10⁶ 40.3 1.8 × 10⁻¹ 4 1 6.9 × 10⁵ 9.6 4.2× 10⁻² 5 1 5.1 × 10⁵ 7.1 3.1 × 10⁻² 6 1 3.9 × 10⁵ 5.4 2.4 × 10⁻² 7 2 1.4× 10⁵ 1.9 8.3 × 10⁻³ 8 2 1.4 × 10⁵ 1.9 8.3 × 10⁻³ 9 2 2.9 × 10⁵ 4.0 1.8× 10⁻² 10 3 1.6 × 10⁵ 2.2 9.6 × 10⁻³ 11 3 1.3 × 10⁵ 1.8 7.9 × 10⁻³ 12 31.8 × 10⁵ 2.5 11.0 × 10⁻³  The results indicate that ¹²⁵I NOCC adheresto rat femur. After a third wash, it was found that 9.5 × 10−3 +/− 0.002μL/mm² (or about 0.1 μg NOCC/mm²) of ¹²⁵I NOCC remained adhered to therat femur.

EXAMPLE 3

This example illustrates the effect of adherent NOCC on cellularattachment. In the first assay, 3T3 fibroblasts were maintained inculture in RPMI culture medium supplemented with 10% fetal calf serum(FCS), 20 mM HEPES, 100 U/ml penicillin/streptomycin, 2 mM 1-glutamine,and 50 μM 2-mercaptoethanol; referred to as complete RPMI (cRPMI). 3T3fibroblasts were removed from the stock culture flask by treatment withtrypsin, washed and re-suspended to a concentration of 3.0×10⁵ cells/mlin serum free RPMI (sfRPMI; RPMI as before but without the 10% FCS)either alone or with 0.1% NOCC.

These cells were then plated on 96 well Nunclon tissue culture plateswhich had been pre-coated (overnight, room temperature) with one of fourdifferent coating treatments. These were: 1) phosphate buffered saline(PBS) as a control, 2) vitronectin at a concentration of 15 μg/ml in PBS(vitronectin is an ECM protein that fibroblasts adhere to), 3) sfRPMI asa control, and 4) cRPMI, which contains many ECM proteins. Afterpre-coating, plates were washed three times in PBS to remove the coatingmedia. Cells were plated at a concentration of 3.0×10⁴ cells/well andincubated at 37° C. for 90 minutes. Cells were then observedmicroscopically, and classed as either adherent or non-adherent based onmorphology. Two hundred cells were counted in each well (each coatingtreatment was done in triplicate), and a mean % adherence±standarddeviation (SD) was calculated.

This second assay allows quantification of the effect of adherent NOCCon fibroblast adhesion. In this assay, 3T3 cells were labeled withradioactive chromium (⁵¹Cr, in the form of Na₂ ⁵¹CrO₄) suspended insfRPMI, added to the wells of 96-well Nunclon delta plastic plates at aconcentration of 2×10⁴ cells/well and allowed to adhere. After a 90minute incubation at 37° C., a large proportion of fibroblasts willadhere to plastic. Washing of the plate with PBS removed non- or looselyadherent cells. The number of remaining adherent cells was assessed bylysis with 10% sodium dodecyl sulfate (SDS; a detergent) and harvestingthe well contents. The lysate was then counted in a gamma counter anddisintegrations per minute were recorded. The level of radioactivityfrom the lysate was compared to that present in 2×10⁴ labeledfibroblasts and is indicative of the number of cells adhering to eachwell. The adhesion assay was performed in the presence or absence of0.1% NOCC.

Visual inspection of fibroblast morphology in the presence or absence ofNOCC, demonstrated that when fibroblasts were plated in sfRPMI, theyconsistently displayed an “adherent” morphology; that is, the cells wereflattened with complex processes. Regardless of the coating treatment,approximately 80% of cells observed looked like normal culturedfibroblasts. In contrast, when fibroblasts were plated in sfRPMI with0.1% NOCC, the number of cells displaying the adherent morphology wasgreatly reduced. In fact, wells pre-coated with PBS or sfRPMI (no ECMproteins present), no cells adhered in the presence of NOCC. When wellswere pre-coated with vitronectin or cRPMI, some cells adhered but theadherence was significantly less than that which occurred in the absenceof NOCC (FIG. 7).

Hyaluronic acid (HA) was also tested in this system, to determinewhether it had similar effects to NOCC. When a similar morphologicalexamination was performed on cells plated in sfRPMI containing 0.1% HA,it was observed that the HA did not have the same effect on fibroblastmorphology (FIG. 8).

The ⁵¹Cr adhesion assay was developed to achieve a more quantitativemethod of measuring fibroblast adherence. The first experiment using the⁵¹Cr adhesion assay confirmed the results obtained by visual inspectionexamining the effect of adherent NOCC on adhesion of 3T3 fibroblasts touncoated Nunclon delta plates. The results confirmed that adherent NOCCblocks adhesion of 3T3 fibroblasts to plastic, by more than 90% usingthis assay (FIG. 9). This result, taken together with the previous work,suggests that NOCC adheres to the plastic and interferes with thedeposition of ECM proteins in a competitive manner.

A competitive assay was performed to test these results with varyingconcentrations of FCS, which contains the ECM proteins of interest. The⁵¹Cr adhesion assay was performed using RPMI supplemented with 2%, 5% or10% FCS as a plating medium (in the presence or absence of 0.1% NOCC).It was found that presence of FCS in the plating medium reversed theinhibitory effect of adherent NOCC on fibroblast adhesion in a dosedependent manner, where 10% FCS fully restored binding of 3T3 to theplates in the presence of adherent NOCC (FIG. 10). There were however,two possible explanations for this effect: 1) adherent NOCC preventsfibroblast adhesion to the ECM proteins which bind to the plate, or 2)adherent NOCC prevents the binding of ECM proteins to the plate in acompetitive manner.

To address these possibilities, plates were pre-coated with ECM in theform of RPMI containing varying concentrations of FCS ranging from 2% to10% overnight at 4° C. The unbound ECM proteins were then washed off.The adhesion assay was performed using these pre-coated plates and cellssuspended in sfRPMI in the presence or absence of 0.1% NOCC. Thepresence of adherent NOCC did not interfere with the adhesion offibroblasts to the coated plates, thus confirming that adherent NOCCdoes not inhibit adhesion of fibroblasts to ECM proteins alreadydeposited on the plate (FIG. 11).

To confirm that adherent NOCC competitively interferes with depositionof ECM proteins on plastic surface, plates were pre-coated with NOCC (insfRPMI) overnight at 4° C., and washed. Fibroblasts suspended in RPMIsupplemented with 2%, 5% or 10% FCS were allowed to adhere to such NOCCcoated plates or control uncoated plates. In this experiment, theadherence of fibroblasts was determined after 1 hour by measuring theactivity of the tritiated thymidine-labelled cells that were attached tothe plates. The results (—FIG. 12) showed that fibroblast adherence toNOCC coated plastic is eliminated in the presence of 2, 5 or 10% FCS,supporting the hypothesis that adherent NOCC binds to the plastic platesurface and prevents the deposition of ECM proteins. In the absence ofECM network, fibroblasts are unable to bind to the substrate.

EXAMPLE 4

In this Example, epithethial cells labeled with tritiated thymidine wereused to test whether NOCC could eliminate their attachment to cultureplates. Culture plates were pre-coated with NOCC (in sfRPMI) overnightat 4° C., and washed. Labeled epithial cells were suspended in RPMIwhich was supplemented with FCS and were allowed to adhere to the NOCCcoated plates or control uncoated plates. The adherence of theepithelial cells was determined after 1 hour by measuring the activityof the tritiated thymidine-labelled cells following lysis from theplates. The results (shown in FIG. 13) show that the NOCC pre-coatingprevents the attachment of epithelial cells

EXAMPLE 5

In this Example, an experiment similar to that described in Example 4was carried out except instead of labeled epitheial cells, tritiatedthymidine labeled macrophages (J774M0) were used. Again, the plates wereprecoated with 0.1% NOCC, and the macrophages were suspended in RPMI andwere allowed to adhere to the NOCC coated plates or control uncoatedplates. FIG. 14 illustrates that the NOCC precoating inhibits theattachment of the macrophages to the plates.

The foregoing examples are merely exemplary and those skilled in the artwill be able to determine other modifications to the describedprocedures which fall within the scope of the invention. Accordingly,the invention is defined by the following claims and equivalentsthereof.

1. A method of inhibiting attachment of a protein to a substratecomprising applying an adherent N, O-carboxymethylchitosan coatingselected from the group consisting of ionically cross-linked andcovalently crosslinked N, O-carboxymethylchitosan to said substrate suchthat such attachment of said protein is inhibited, and provided that thesubstrate is not mammalian or non-mammalian cells or tissue whereinadhesion is being inhibited.
 2. The method of claim 1, wherein thesubstrate is selected from medical devices, fermentation units,bioreactors, and solid supports.
 3. The method of claim 2, wherein themammalian tissue is soft tissue.
 4. The method of claim 2, wherein themammalian tissue is hard tissue.
 5. The method of claim 2, wherein saidmedical device is selected from the group consisting of stents,catheters, contact lenses, breast implants, pacemakers and shunts. 6.The method of claim 1, wherein said adherent N,O-carboxymethylchitosanis an adherent solution, hydrogel, paste, particle, bead, pellet,rehydratable film, or sponge.
 7. A method of obtaining a population ofcells comprising: a) supplementing culture media with an adherentN,O-carboxymethylchitosan selected from the group consisting ofionically cross-linked and covalently cross-linkedN,O-carboxymethylchitosan; b) growing said population of cells in thesupplemented media; and c) allowing said cells to grow or differentiate,wherein proliferation of substrate-dependent cells within saidpopulation is inhibited.
 8. The method of claim 7, wherein said adherentN,O-carboxymethylchitosan is a solution, hydrogel or paste.
 9. Themethod of claim 7, wherein substrate-dependent cell attachment withinsaid population is inhibited.